The present invention relates to the method for reproducing coniferous plants by somatic embryogenesis using the techniques of plant tissue culture. It is especially directed to production of totipotent embryonal cells in high concentrations relative to associated nonembryogenic cells. These cells are particularly useful for processes involving genetic manipulation and high frequency recovery of genetically transformed plantlets.
Loblolly pine (Pinus taeda L.), its closely related southern pines, and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) are probably the most important commercial species of temperate North American timber tress. Similarly, Norway spruce (Picea abies (L.) Karst. ) is probably the most important European softwood species. Since the early 1940s, when serious private reforestation efforts began, literally billions of one and two year old nursery-grown trees have been planted worldwide on cut-over or burned forest lands. For many years these seedling trees were grown using naturally produced seed from cones collected as a part time effort of individuals seeking to supplement their incomes. As early as 1957 forest geneticists began to plant seed orchards using either seed or grafted scions obtained from superior trees. These trees were selected for such inheritable characteristics as rapid growth, straightness of bole, wood density, etc. Now in both the southern pine and Douglas-fir regions the bulk of the seed is produced from selected trees grown in seed orchards, some of them now second and third generation orchards.
Despite the fact that the orchards were stocked with superior trees, pollination often cannot be carefully controlled and frequently the seed trees are fertilized by wild pollen of unknown characteristics. For this reason, the characteristics of the progeny produced by sexual reproduction have not been as predictable as hoped and genetic gain could not be attained as rapidly as desired.
Beginning about 1960, techniques were developed for reproducting some species of plants by tissue culture. These were predominately angiosperms and usually ornamental house plants. The method employed use of a suitable explant or donor tissue from a desirable plant. This was placed on a series of culture media in which nutrients and growth hormones were carefully controlled from step to step. The usual progression was growth from the explant to a callus. The callus was placed on a budding medium where adventitious buds formed. These, in turn, were separated, elongated, and rooted to ultimately form plantlets. A plantlet has the nature of a seedling but is genetically identical to the explant donor plant.
Gymnosperms in general, and most forest tree species in particular, proved to be much more difficult to reproduce by tissue culture. It was not until about 1975 that Douglas-fir was successfully reproduced by organogenesis. Loblolly pine was successfully reproduced about two years later.
Culture by organogenesis is tedious and expensive due to the large amount of delicate manual handling necessary. It was soon recognized that embryogenesis was potentially a much more desirable method from the standpoints of quantity of plantlets produced, cost, and potential genetic gain. Work on embryogenesis of forest species began in the late 1970s. U.S. Pat. No. 4,217,730 to El-Nil describes one early attempt at somatic embryogenesis of Douglas-fir. This approach was later set aside because advanced stage embryos and plantlets could not be readily obtained. However, other workers entered the field in increasing numbers and progress has been rapid even if it has not until the present time reached the commercial stage. A brief review of some of the most important work will follow. This is intended to be representative and is not fully inclusive of all the work in the field. Literature citations in the text are given in abbreviated form. Reference should be made to the bibliography at the end of the specification for full citations of the literature noted herein.
The natural embryogeny of gymnosperms is described in great detail by Singh (1978). Conifer-type embryogeny is one of four types noted for gymnosperms. This includes virtually all of the important forest species except Sequoia. Singh notes that the immature seeds typically contain more than one embryo. Most commonly this seems to occur when a single zygote forms multiple embryos, a phenomenon called "cleavage polyembryony". As the seed matures one embryo becomes dominant while the others are suppressed. The ability to form multiple embryos from a single zygote forms the basis for most of the present embryogenic processes for multiplying conifers. However, Douglas-fir is an exception. Most typically only a single embryo will be present throughout the formation and maturation of a seed. This may account for at least some of the difficulty experienced to date in multiplying Douglas-fir by somatic embryogenesis.
Bourgkard and Favre (1988) describe what is the apparently successful production of plantlets by somatic embryogenesis of Sequoia sempervirens. As a historic note, this was one of the first forest tree species successfully reproduced by organogenesis.
Hakman and her coworkers have concentrated on Norway spruce (Picea abies), apparently with some success. In a paper by Hakman, Fowke, von Arnold, and Eriksson (1985) the authors describe the production of "embryos" but not plantlets. Hakman and von Arnold (1985) do suggest that they have successfully obtained plantlets. This latter paper is interesting for its comments on the variability within the species and the poor success with many of the seed sources used for explants. The authors suggest that this variability may be due to the physiological condition of the source material. However, other workers have noted great differences in behaviour between recognized genotypes of the species.
Nagmani and Bonga (1985) describe embryogenesis from megagametophytes of Larix decidua by tissue culture. The archegonia, proembryos, or embryos with their suspensors were removed prior to culture. Some of the resulting embryos produced in culture were stated to have further advanced to become plantlets established in soil. The ploidy of these plants was not investigated.
Successful production of small quantities of plantlets has now been reported for loblolly pine. Teasdale, Dawson, and Woolhouse (1986) showed the criticality of proper mineral nutrients for cell suspension cultures of loblolly pine. The article by Becwar, Wann, and Nagmani (1988) is enlightening for the differences shown in performance between different families (or genotypes). Three families out of the ten tried accounted for most of their success. Even so, they appeared unable to grow cotyledonary embryos. A companion paper by Nagmani and Becwar (1988) showed development of Pinus taeda to the precotyledonary stage. In an earlier paper, Gupta and Durzan (1987a) described their success in taking loblolly pine to the plantlet stage by embryogenesis. However, only one genotype was successfully taken to the plantlet stage and only one converted plant was produced. The authors note the need for "improved conversion rates" as well as other information before the process can be considered commercially practical.
Sugar pine (Pinus lambertiana) has also been cultured to the plantlet stage as reported by Gupta and Durzan (1986). The authors note a very low 1-2% conversion of embryos into plantlets.
The above researchers appear to be the only ones who have previously reported success in producing Douglas-fir plantlets by somatic embryogenesis (Durzan and Gupta 1987). Again, the success ratio appears to be very low and they have obtained only two converted plants from a single genotype.
In our parent to the present application, Ser. No. 321,035, filed Mar. 9, 1989, now U.S. Pat. No. 4,957,866 herein incorporated by reference, we described an improved method for reproducing coniferous species by somatic embryo-genesis. An intermediate high osmoticant culture medium was used to generate strong late stage proembryos, prior to the development of mature cotyledonary embryos in another medium containing abscisic acid. This step led to a very significantly improved success rate in attaining robust somatic embryos that could be converted into growing plants.
Genetic manipulation has been used on numbers of plant species to introduce naturally lacking characteristics, such as resistance to specific diseases. The use of these techniques is in its infancy, as far as forest tree species are concerned, even though they could prove to be an extremely important part of a tree improvement program.
Removal of cell walls to create protoplasts opens one route to genetic modification. Gene transfer can be accomplished using such techniques as electroporation, protoplast fusion, and DNA uptake. Early work along these lines has not been so much concerned with genetic modification per se as with the development of techniques to obtain viable regenerated cells for further development. A number of examples can be cited. These papers are typical of the work reported to date and are not offered as an exhaustive survey of the literature on conifer protoplasts. Gupta and Durzan (1987b) showed that protoplasts can be formed from very early somatic embryos of loblolly pine. Cell walls were successfully regenerated on these protoplasts and culture was resumed so that somatic embryos were ultimately obtained. Successful conversion to plants was not reported.
Attree and coworkers (1989) report regeneration of white spruce (Picea glauca (Moench) Voss) protoplasts which had been isolated from cryopreserved embryogenic tissue. Cotyledonary embryos formed from the regenerated protoplasts were ultimately successfully germinated, although the authors do not indicate whether any of the germinants were ultimately converted to plants growing in soil.
Tautorus, et al. (1990) similarly describe the production and regeneration of protoplasts of black spruce (Picea mariana Mill.). These authors did not report what progress they had made in achieving germination of somatic embryos.
The efforts noted above are pioneering from the standpoint that they explore one route to ultimate genetic manipulation of conifers. However, many other routes are available and the general methods that can be employed are well known.
The techniques of somatic embryogenesis just discussed appear to provide a particularly useful tool to use in the genetic modification of conifers. However, one problem was not discussed or perhaps even considered by any of the above workers. This is the matter of obtaining a more suitable germ plasm supply for attempted genetic modification. Many, if not most, of the cells present among those available to the present time lack the potential of ultimately developing into embryos useful for growing plants. In addition to the embryonal cells, there is a large volume of attached and free suspensor cells and other nonembryogenic material that, in effect, serves as a very significant diluent. This greatly reduces the statistical probability of a useful modification occurring in a cell that can later be regenerated and further processed to result in a growing plant.
The present invention is directed to the problem of obtaining the high quantities and concentrations of embryonal cells needed for genetic manipulation, by whatever method is chosen, and the high frequency recovery of genetically transformed plantlets.